Insulin is a protein and more specifically a hormone that controls the metabolism of glucose. Lack of insulin within an animal results in the animal developing diabetes and excess amounts of insulin results in coma. Insulin is a polypeptide which is produced by the betacells of the islets of Langerhans of the pancreas. Pancreatic secretions of insulin are stimulated by high blood levels of glucose and amino acids after meals. Glucose uptake is then stimulated by the action of insulin on various tissues (e.g., muscles, liver, and fat). Insulin also stimulates glycogen and fat synthesis. Pharmaceutical preparations of insulin are used therapeutically in the treatment of diabetes mellitus known as type I and type II diabetes.
The inability of certain animals, such as humans, to generate sufficient amounts of insulin in the pancreas leads to the development of diabetes mellitus. Diabetes mellitus is a syndrome characterized by abnormal insulin secretion and various metabolic and vascular impairments. Individuals suffering from diabetes mellitus are currently treated by the administration of porcine, Dovine or recombinant human insulin. The administration of insulin, however, does not consistently mimic the effects of endogenous insulin and may result in hypoglycemia and long-term complications such as atherosclerosis.
Animal derived insulin is not chemically identical to human insulin and sometimes contains other biologically active impurities. Efforts were made to develop a means of producing insulin which is chemically identical to human insulin and which does not include any biologically active impurities. As a result of such efforts, recombinant techniques have been developed to produce human insulin and proinsulin polypeptides in microorganisms which are isolated and purified for pharmaceutical use. For example, see European Patent Application No. 0 055 945, published 14 Jul. 1982, which application is incorporated herein by reference, for its disclosure of recombinant techniques and methodologies for producing insulin and proinsulin polypeptides and the isolation of such polypeptides.
As indicated above, present technology makes it possible to recombinantly produce insulin having an identical amino acid sequence to human proinsulin and/or insulin which can then be purified and used in pharmaceutical preparations. However, insulin has a plurality of functions. For example, it inhibits hepatic glucose production and stimulates peripheral glucose utilization thereby controlling the metabolism of glucose. Because of these different functions and because the site of administration of the exogenous insulin typically is different from the natural release site, when individuals suffering from diabetes are treated it may be desirable if the insulin which is administered inhibited hepatic glucose production to a greater degree compared with its ability to stimulate peripheral glucose utilization. The need for the insulin administered to act differently from natural insulin in order to obtain a more natural result is explained further below.
Insulin has an overall effect of lowering the glucose concentration in the bloodstream. This effect is obtained by the operation of insulin on two different types of tissue.
The glucose lowering effects of insulin occur in both hepatic and peripheral tissues in order to regulate glucose levels in blood. The interaction of insulin with hepatic receptors results in a decrease in glucose production by the liver as well as increased liver storage of glucose as glycogen; interaction with the peripheral receptors on fat and muscle cells results in an increase in glucose utilization. Both interactions result in a lowering of glucose concentration in the bloodstream. In both liver and peripheral cells, binding to the receptor is concomitant with insulin clearance from the system; i.e., as insulin is utilized, it is also cleared.
In the normal operation of endogenous insulin, the majority of the hormone secreted by the pancreas interacts with the hepatic receptors. This apparent preference is thought to be due to the proximity of the liver to the source of the hormone. Once released into the general circulation, most of the insulin appears to be utilized by peripheral cells, due to the large number of peripheral receptors available. One reason why the administration of insulin does not achieve the "natural" balance between hepatic and peripheral activity may be that the initial introduction of the drug into the general circulation system provides little opportunity for interaction with the hepatic receptors. However, a high degree of such interaction takes place when endogenous insulin is released from the pancreas.
Insulin is initially synthesized in the islets of Langerhans of the pancreas as the single chain peptide proinsulin. Proinsulin has 1-2% of the potency of native insulin when assayed in vitro, about 15% of the potency of insulin in vivo, and a circulating half-life of 30 min as compared to 4 min for insulin. Proinsulin is relatively hepatoselective in vivo (Glauber, H. S., et al., Diabetes (1986) 35:311-317; Peary, D. E., et al., J, Biol. Chem. (1985) 260:13989-13994; Davis, S. N., et al., Diabetes (1988) 37:74 (abstract)). The in vivo hepatoselectivity of proinsulin is 50% more than that of insulin per se. However, proinsulin has too low a potency for most uses.
Insulin is thought to circulate predominantly as a monomer. The monomer is a disulfide-linked, two-chain molecule consisting of A chain of 21 amino acids and B chain of 30. The amino acid sequences of human, porcine and bovine insulin are well established. (The amino acid sequences of insulins of many other species have also been determined.) Attempts to discern which are the essential residues in these peptides were begun some time ago. By observing the conservation of residues between the insulins derived from various species, it was suggested that a largely invariable region on the surface of the monomer is the receptor binding region. This region includes A-chain residues A1 (Gly), A5 (Gln), A19 (Tyr), and A21 (Asn) as well as B chain residues B24 (Phe), B25 (Phe), B26 (Tyr), B12 (Val), and B16 (Tyr).
de Meyts, R. A., et al., Nature (1978) 273:504-509, tested 29 insulin-type molecules including animal insulins and proinsulins, insulin-like growth factors and chemically modified insulins for ability to bind to receptor and for biological potency. de Meyts et al. found a one thousand-fold variation over the series of 29 analogs wherein the essential residues were shown to be some of the 8 carboxyterminal residues of the B chain and the A21 (Ash) residue of the A chain.
Tompkins, C. B., et al., Diabetologia (1981) 20:94-101, showed that certain analogs stimulated hypoglycemia entirely by increasing peripheral glucose uptake, whereas others did so by decreasing hepatic glucose production. In these studies, A1, B29-diacetyl derivatives of insulin were able to stimulate peripheral glucose uptake, while A1-B29 cross-linked insulins and proinsulin decreased hepatic glucose production.
Later studies by Nakagawa, S. H., et al., J. Biol, Chem (1986) 261:7332-7341, confirmed the importance of the carboxy terminal region of the B chain. Studies of binding to the hepatocyte receptor showed that insulin residues B26-B30 could be deleted without decrease in binding or biological potency when the carboxyl group is alphacarboxamidated to preserve the hydrophobic character of the carboxy terminal B chain domain. However, deletion of residues B25-B30 or B24-B30 resulted in a decrease in potency.
A reduction in potency was also observed when the phenylalanine at B25 was replaced by leu or ser or by homophenylalanine; however, replacement by naphthyl-1-alanine or naphthyl-2-alanine at B25 decreased binding activity to a lesser extent. The decreased activity effected by replacement of Phe at B25 by Ala, Ser, Leu or homophenylalanine was reversible by deletion of the remaining carboxy-terminal residues B26-B30. The authors concluded that steric hindrance involving the carboxy-terminal domain of the B chain helped direct interaction of insulin with its receptor, that the negative effect of this domain is "reversed by filling of a site reflecting interaction of the receptor and the beta-aromatic ring of B25 (phe)" and that the remaining carboxy-terminal residues, besides B25, were important in effecting the interaction of this residue with the receptor. Further studies by this group (Nakagawa, S. H., et al., J. Biol. Chem (1987) 262:1254-1258) showed that the downstream residues must be deleted to reverse the effect of replacement of B25 (Phe) by ser and that replacement of residues B26 (Tyr) or B27 (Thr) does not reverse this decrease in affinity. It was further shown that cross-linking between B29 (Lys) and A 1 (Gly) decreases the affinity of insulin for the receptor. These studies were directed to an effort to enhance the potency of insulin.
Coincidentally, however, a diabetic patient was shown to produce a mutant form of insulin having Leu instead of Phe at B25. Two other patients were shown to have mutations at the codons for B24 or B25, but the encoded insulin was not characterized (Shoelson, S., et al., Nature (1983) 302:540-543).
Increased binding to receptor and increased potency was shown to be a property of insulin iodinated at the tyrosine residue B26 (Podlecki, D. A., et al., Diabetes (1983) 32:697-704). Similarly, Schwartz, G. P., et al., Proc. Natl. Acad. Sci. USA (1987) 84:6408-6411, described a superactive insulin with enhanced binding both to hepatic and peripheral receptors which contains an aspartic acid substitution for the natural histidine at B10 of human insulin.
Still others have reported insulin analogs which have specified properties thought desirable. For example, Brange, J., et al., Nature (1988) 333:679-682, prepared analogs with substitutions at B9, B12, BlO, B26, B27 and B28 which are designed to prevent formation of dimers.
International Patent Application WO 90/12814, published 1 Nov. 1990, discloses hepato-specific insulin analogs wherein tryptophan or other bulky, hydrophobic residues selected from the group consisting of tryptophan, naphthylalanine, N-gamma-dansyl-alpha, gamma-diaminobutyric acid, leucine, valine, phenylalanine and other hydrophobic amino acids are substituted at the A13, A14, A15, A19 and B16 positions of the insulin polypeptide. The naturally occurring amino acids which are conventionally described as hydrophobic are alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine and tryptophan, or alternatively amino acids whose side chains consist only of hydrocarbon, except for the sulfur atom of methionine and the nitrogen atom of tryptophan (J- Darnell, Molecular Cell Biology, Scientific American Books, New York, 1986). In contrast, the sidechains of histidine and glutamine are described as polar or hydrophilic groups.
Contrary to the teaching in WO 90/12814, the present invention provides the surprising and beneficial result that substitution by non-hydrophobic amino acids at A19 produces insulin analogs which are hepatoselective in vivo. Further, the present invention provides the surprising and beneficial result that substitution by phenylalanine at A14 produces analogs that are peripheral selective. Such A14 analogs have a different and distinct therapeutic use from the hepatospecific use disclosed in International Patent Application WO 90/12814.
The present invention recognizes the desirability of tissue selectivity when providing insulin to a patient from an exogenous source, i.e., not directly from the pancreas.